Affinity chromatography is a separation and purification technique that is based upon a unique and fundamental biological property of biological molecules, namely their selective, specific high-affinity recognition of and reversible molecular interaction with other molecules. Most biological macromolecules have a substrate or functional binding site for a specific ligand or an effector molecule, which itself may be another protein.
In general, a specific ligand is covalently attached to a solid support matrix. A sample containing the biological molecule which will specifically bind (absorb) to the immobilized ligand is brought into contact with the immobilized ligand. After unabsorbed and contaminating molecules are removed, the specifically bound molecule is eluted from the solid support by disrupting the specifically bound molecule-ligand interaction by one of several procedures, such as by changing the ionic strength or pH of elution buffers.
By this procedure, immobilized drugs, vitamins, peptides, hormones and the like may be used to isolate corresponding receptors or transport proteins. Immobilized protein can serve to isolate other complementary or interacting proteins. Similarly, such a procedure can be used to separate particulate biological specimens, such as cell membranes and even intact cells bearing specific receptors. Use of such a procedure is also useful to purify polynucleotides, antigens, antibodies, virus, enzymes and the like. In addition, such solid based affinity support matrixes have been utilized to immobilize enzymes for use in reactions as catalysts and the like.
Heretofore, the most widely used solid matrixes for affinity chromatography have been polysaccharide based matrixes, such as agarose, cellulose and crosslinked dextrin, although crosslinked polyacrylamides and silica or microporous glass beads have also been used.
However, with most such solid matrixes and especially with silica based solid matrixes, the solid base material is not sufficiently shielded from the molecules in the biological sample of interest with the result that non-specific binding, in the nature of absorption with materials other than the specific binding partner of the immobilized ligand, occurs. With the silica based solid matrixes heretofore utilized strong hydrogen bonding and absorption at silanol groups occurs and is disruptive of the process. Moreover, the aqueous solutions that must be employed with the proteins or other biological material of interest cause undesirable hydrolysis of the silica matrixes, leaching undesirable material from the matrixes, resulting in unstable matrixes.
With the heretofore used solid support matrixes employed in affinity chromatography one could generally only use such supports for about 200 to 300 hours in aqueous solutions and even less in acid or alkaline pH environments. Furthermore, such prior art matrixes could not tolerate any exposure to 0.1N NaOH.
Thus, the development of affinity chromatography has been inhibited to a great extent by these drawbacks of the presently used solid support matrixes, particularly due to the absorption of non-specific materials and instability of the matrixes.
It is therefore desirable that solid support matrixes for affinity chromatography be provided which eliminate or substantially reduce the risk of non-specific binding and which are stable under a variety of pH conditions and in aqueous solutions.